Cross-species RNAi therapy via AAV delivery alleviates neuropathic pain by targeting GCH1.

Tetrahydrobiopterin (BH4) expression is normally strictly controlled; however, its intracellular levels increase considerably following nerve damage. GTP cyclohydrolase I (GCH1) plays a crucial role in regulating BH4 concentration, with an upregulation observed in the dorsal root ganglion in cases of neuropathic pain. In this study, we aimed to develop and evaluate the clinical potential of an RNA interference-based adeno-associated virus (AAV) targeting GCH1 across various species to decrease BH4 levels and, consequently, alleviate neuropathic pain symptoms. We identified universal small-interfering RNA sequences effective across species and developed an AAV-u-shRNA that successfully suppressed GCH1 expression with minimal off-target effects. Male Sprague Dawley rats were divided into four groups: normal, spared nerve injury, AAV-shCON, and AAV-u-shGCH1. The rats were sacrificed on post-injection day 28 to collect blood for BH4 level assessment. The AAV-u-shGCH1 group demonstrated remarkable improvement in the mechanical withdrawal threshold by PID 28, significantly outperforming the normal, spared nerve injury, and AAV-shCON groups. Plasma BH4 levels confirmed that AAV-u-shGCH1 effectively reduced neuropathic pain by inhibiting BH4 synthesis in vivo, introducing a novel, multispecies-compatible therapeutic strategy. Our results suggest that a single application of AAV-u-shGCH1 could offer a viable solution for neuropathic pain relief.